hello

International Journal of Food Microbiology 167 (2013) 2–7

Contents lists available at ScienceDirect

International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Principles for the risk assessment of genetically modified microorganisms and their food products in the European Union☆

Jaime Aguilera ⁎, Ana R. Gomes, Irina Olaru European Food Safety Authority, Via Carlo Magno 1/a, 43126 Parma, Italy

☆ The views or positions expressed in this article corresp and cannot be regarded as representing the position, the ⁎ Corresponding author. Tel.: +39 0521036531.

E-mail address: [email protected] (J. Ag

0168-1605/$ – see front matter © 2013 Elsevier B.V. Al http://dx.doi.org/10.1016/j.ijfoodmicro.2013.03.013

a b s t r a c t

a r t i c l e i n f o

Available online 23 March 2013

Keywords: Genetically modified organisms Genetically modified microorganisms Food safety Risk assessment Guidance Regulation

Genetically modified microorganisms (GMMs) are involved in the production of a variety of food and feed. The release and consumption of these products can raise questions about health and environmental safety. Therefore, the European Union has different legislative instruments in place in order to ensure the safety of such products. A key requirement is to conduct a scientific risk assessment as a prerequisite for the product to be placed on the market. This risk assessment is performed by the European Food Safety Authority (EFSA), through its Scientific Panels. The EFSA Panel on Genetically Modified Organisms has published complete and comprehensive guidance for the risk assessment of GMMs and their products for food and/or feed use, in which the strategy and the criteria to conduct the assessment are explained, as well as the scientific data to be provided in applications for regulated products. This Guidance follows the main risk assessment principles developed by various international organisations (Codex Alimentarius, 2003; OECD, 2010). The assessment considers two as- pects: the characterisation of the GMM and the possible effects of its modification with respect to safety, and the safety of the product itself. Due to the existing diversity of GMMs and their products, a categorisation is recommended to optimise the assessment and to determine the extent of the required data. The assessment starts with a comprehensive characterisation of the GMM, covering the recipient/parental organism, the donor(s) of the genetic material, the genetic modification, and the final GMM and its phenotype. Evaluation of the compo- sition, potential toxicity and/or allergenicity, nutritional value and environmental impact of the product constitute further cornerstones of the process. The outcome of the assessment is reflected in a scientific opinion which indi- cates whether the product raises any safety issues. This opinion is taken into account by the different European regulatory authorities prior to a decision regarding authorisation to commercialise the product.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Microorganisms have been the source of food and food products from the dawn of civilisation. With the advent of the recombinant DNA technology, the use of microorganisms as biofactories for the synthesis of new products had a boost. Currently, the use of genetically modified microorganisms (GMMs) in the food industry is well established. Esti- mations from 2009 indicate that around 40% of the food enzymes marketed in Europe are produced by recombinant strains, both bacterial and fungal (AMFEP, 2009). Food applications of GMMs go far beyond en- zymes. Additives such as vitamins, amino acids, and polysaccharides can also be obtained from recombinant strains. The future commercial land- scape of GMMs in food will also likely include biomass-derived products, and even whole GMMs. There are developments on food supplements made from GM microalgae with an enriched oil content (Franklin et al.,

ond to the authors and are not, views or the policy of EFSA.

uilera).

l rights reserved.

2011). Recombinant baker's yeasts with enhanced fitness for industrial baking are not a novelty (Prieto et al., 2006), and there are already GM yeast for wine-making purposes commercially available in the USA and Canada (FDA, 2003). Possible future uses of GMMs in foods extend to dairy products such as yogurt or cheese.

2. Regulatory framework

Food and feed consisting of, containing, or derived from genetically modified organisms (GMOs, mainly GM grain crops) are circulating on the world markets for more than a decade and, as for other fields of the food and feed industry, the need for a safety evaluation of such prod- ucts (to be introduced or already existing on the market) is largely agreed. In fact, international standards on the safety of GMOs, including GMMs, have been developed (Codex Alimentarius, 2003; OECD, 2010), and the safety of GMM-produced food enzymes has been reviewed else- where (Pariza and Johnson, 2001; Olempska-Beer et al., 2006). Regula- tion of GMOs exists in many countries. In the European Union, there are several legislations in place to ensure that GM food products are safe for humans, animals and the environment. Attending to the nature

3J. Aguilera et al. / International Journal of Food Microbiology 167 (2013) 2–7

of the product, food and food products obtained with the use of GMMs would fall under different legislative pieces:

• Food enzymes, food additives and food flavourings. These products, whatever their origin, fall under either Regulation (EC) No 1332/ 2008 (EC, 2008a) on food enzymes, Regulation (EC) No 1333/2008 (EC, 2008b) on food additives, or Regulation (EC) No 1334/2008 (EC, 2008c) on food flavourings, respectively. Regulation (EC) No 1331/2008 (EC, 2008d) on the common authorisation procedure applies to the three types of substances. When the product is obtained by fermentation of a GMM, these regulations apply if the GMM has been totally removed.1 No particular authorisation or labelling is fore- seen other than those already foreseen for any product under these regulations. However, the safety aspects of the genetic modification of the production strain are taken into account in the risk assessment (see below).

• Products derived from biomass of a GMM (i.e. containing rests of cells of the GMM, such as biomass-based food supplements or bread) fall under Regulation (EC) No 1829/2003 on genetically modified food and feed (EC, 2003). This regulation applies to food and feed consisting of, containing or derived from GMOs, and covers food and feed safety issues, as well as environmental risks potentially posed by such food and feed. Food enzymes, food additives and food flavourings produced by GMMs also fall under this Regulation if the GMM has not been totally removed from the product.

• Products consisting of or containing GMMs (i.e. the GMM is not destroyed or inactivated, such as yeast for baking, yogurt, and starter cultures for cheese) also fall under Regulation (EC) No 1829/2003. In addition, as the GMM is still viable, they fall under Directive 2001/ 18/EC on the deliberate release into the environment of GMOs (EC, 2001). This Directive covers environmental safety issues resulting from the release of GMOs, and establishes the principles and structure to articulate an environmental risk assessment (ERA). By virtue of a mechanism known as “one door-one key”, a single application under the Regulation, containing all the data covering food and environmen- tal safety of the product, can be submitted to request authorisation under both the Regulation and the Directive.

Although each of the above-mentioned legislations is independent and covers the particularities of the different products under their scope, they share some essential principles relative to safety and mar- keting. Products must be safe for humans, animals and/or the environ- ment. For the product to be put into the EU market, an authorisation is required and, as a prerequisite to gain the authorisation, a risk assess- ment of the product must be done, which enables to conclude on its safety. Those who intend to commercialise a product must prepare an application, which contains all the necessary scientific information to conduct the risk assessment. This application is forwarded to the European Food Safety Authority (EFSA), which performs an indepen- dent evaluation of the application. EFSA can request from applicants additional information, including experimental data, if deemed neces- sary for the risk assessment. In addition, EFSA takes into account other relevant data, such as peer-reviewed scientific publications. After its evaluation, EFSA issues a scientific opinion on the safety of the product in question. This opinion is considered by the European Commission which, assisted by the Member States, decides on whether to autho- rise or not authorise the product.

In order to provide guidance on how the risk assessment should be conducted, and to help applicants to prepare applications, EFSA has released a series of Guidance Documents, which set out the meth- odology for the safety evaluation, and indicate the data requirements for applications for different products. Documents exist covering food

1 Such products are, from a legal point of view, considered as made with GMMs, in contrast to products in which material derived from the GMMs is still present, that are considered made from GMMs (EC, 2003).

enzymes, flavourings, additives, and GMMs. For the latter, the rele- vant document is the Guidance on the risk assessment of genetically modified microorganisms and their products intended for food and feed use (EFSA, 2011a, hereafter the “GMM Guidance”). This document fully covers the risk assessment of GMMs and food and feed products consisting of, containing, or derived from GMMs (and therefore fall- ing under Regulation (EC) No 1829/2003). In case of enzymes and other fermentation products made with GMMs, the guidance covers the safety aspects related to the genetic modification of the GMM production strain. For the evaluation of the product itself, it provides cross-references to other applicable guidance from EFSA or other sources. Out of the scope of the GMM Guidance are tissue cultures of plant or animal cells, viruses or viroids, GMMs used as plant protection products or biocides, or GMMs released for experimental purposes and for research.

3. Principles of GMM risk assessment and categorisation of products

The general principles of the risk assessment of GMOs are outlined in Directive 2001/18/EC, and reflected in the GMM Guidance. The objective of the risk assessment is to determine the potential adverse effects of the GMM on human and/or animal health and the environ- ment, compared to the non-modified microorganism from which it is derived. Comparison with the non-modified organism is an interna- tionally accepted principle for the assessment of risks derived from GMOs (known as the comparative approach) (Codex Alimentarius, 2003; OECD, 2010). The assessment should also be conducted on a case-by-case basis (each GMM is assessed independently and taking into account its particularities), and should address direct and indi- rect effects, immediate or delayed.

According to the comparative approach, the characteristics of the GMM with potential to cause harm should be identified and com- pared to those presented by the non-modified microorganism (called conventional counterpart) in the context of the intended uses. The conventional counterpart should also have a history of safe use in order to obtain adequate baseline data. The same principle applies for food derived from GMMs: it should be compared with food pro- duced using the traditional microorganisms. Given the wide range of microorganisms used in industrial applications, some of them with a long history of modifications from a wild ancestor (which might not even exist anymore), the ideal situation for a comparative assessment may not always occur. If the conventional counterpart has not a history of safe use, well-known close relatives (e.g. another strain, an ancestor) can be used as additional comparators to obtain reference data, taking into account their relationship and differences. It is also common that the strain which receives the genetic modification has been subject to other genetic modifications in the past, in the context of previous uses. In this case, the recipient strain might be used as comparator if its safety has been established in previous assessments towards a safe non-modified strain. Otherwise, it will be necessary to identify the first wild ancestor and assess all the genetic modifications introduced so far, in addition to the one which is subject to the application. Even so, there might be cases when no adequate comparator can be identified, and therefore the comparative approach is not possible. In those cases, the GMM guidance provides indications on how to conduct a compre- hensive risk assessment, which would include a full toxicological and nutritional assessment of the GMM-derived food (see Sections 4.3.1. and 4.3.3.).

The strategy to be followed for the safety evaluation of the different products covered in the GMM Guidance (including the suitability of the comparative approach) will depend on the nature of each product, which will also determine the extent of the scientific data required. For this reason, products are classified into four categories:

• Category 1. The first category corresponds to chemically defined compounds and their mixtures in which both GMMs and newly

4 J. Aguilera et al. / International Journal of Food Microbiology 167 (2013) 2–7

introduced genes have been removed. Examples are crystallised vitamins or amino acids, and other highly purified molecules. As regards the scope of the GMM Guidance, this category has the least data requirements. Due to its high purity, any potential harm posed by the GMM is not expected to reach the final product, whose safety will be assessed according to the standards for con- ventional products of the same nature.

• Category 2. The second category covers complex products in which both GMMs and newly introduced genes are no longer present. Products can still contain rests of GMM cells. Under this category are the majority of commercial preparations of enzymes. These products are normally obtained by simple separation of the produc- tion strain from the fermentation broth, which is then heated, dried, or subjected to other downstream processing. Therefore, they often contain by-products of fermentation and other impurities. Cell lysates would also fall under Category 2. Provided that both GMMs and newly introduced genes are not present, the assessment of these products will mainly focus on any potential hazardous substance produced as an effect of the genetic modification (including the intended protein or any by-product), which may be present in the final formulation.

• Category 3. Products under this category are those in which viable GMMs are not present, but which still contain recombinant genes. Belonging to this category are biomass-derived food supplements, some beers or cheese. For these products, the risk assessment is extended to the potential changes in the food resulting from the acquired traits of the GMM, either conferred by the introduced DNA or as a result of unintended effects of the genetic modification. In addition, the potential horizontal transfer of the newly introduced genes to other organisms (from the gut or other environments) and its consequences will need to be evaluated.

• Category 4. The most complex level of assessment is reserved to products under Category 4, which encompasses those consisting or containing viable GMMs (e.g. yeast for baking, starter cultures for fermented foods, and yogurt). Here, in addition to all the above, it will be necessary to assess the safety impact of any change in the biology of the GMM with respect to its comparator, in terms of its potential adverse effects to the consumer (toxicity, pathogenicity, effects in the gut microbiota), and to the environment (persistence and effects in the ecosystem).

It is anticipated that most enzymes, additives and other products added to food under the scope of Regulations (EC) No 1331/2008/ to 1334/2008, will fall into Categories 1 and 2, whereas the majority of products under Categories 3 and 4 will be covered by Regulation (EC) No 1829/2003 (Table 1). Because food enzymes, food additives and food flavourings have, according to legislation and guidance, separate criteria for risk assessment, the GMM Guidance does not cover the assessment of those products as such, but provides cross-references to other guidance and relevant documents. The categorisation of the

Table 1 Categorisation of food and food products which can be obtained by the use of GMMs and r

Regulation (EC Regulation (EC Regulation (EC)

Category 1: Chemically defined purified compounds and their mixtures in which both GMMs and newly introduced genes have been removed

Crystallised vit Crystallised am

Category 2: Complex products in which both GMMs and newly introduced genes are no longer present

Food enzyme p

Category 3: Products derived from GMMs in which GMMs capable of multiplication or of transferring genes are not present, but in which newly introduced genes are still present

–

Category 4: Products consisting of or containing GMMs capable of multiplication or of transferring genes

–

a Examples are indicative. Applicants should refer to the European Commission, who is t

product is not left to the decision of the applicant. The GMM Guidance provides clear indications on how to allocate the product into the correct category, the questions to be answered and the experimental evidence to be provided (Table 2, text below).

4. Information requirements for the food/feed safety assessment

The scientific information to be provided in applications must be sufficient to reach a conclusion on whether the product poses any risk(s), and should include experimental evidence obtained from ad-hoc studies, as well as relevant scientific information from available peer-reviewed literature.

4.1. Microbiological and genetic considerations

It is necessary to provide a full characterisation of the GMM, starting from the strain which receives the genetic modification. All the genetic and physiological characteristics need to be described, focusing on the aspects relevant for safety (presence of indigenous mobile elements, capacity for horizontal gene transfer, toxicity, pathogenicity). If the recipient strain has the QPS status (qualified presumption of safety; EFSA, 2007), the information to be provided on its safety can be less extensive. However, it is necessary to unequivocally determine the tax- onomy of the strain, in order to establish that it has a recognised QPS. As pointed out above, if the recipient has been subjected to previous genetic modifications, these should be described in detail. The inserted sequence also needs to be characterised. The GMM guidance recognises sequences from three possible origins: DNA from defined organisms, DNA obtained synthetically, and nucleic acids isolated from environ- mental samples. In all cases, the sequence and function of all the ele- ments of the insert and the vector used for transformation must be described, as well as any artificial mutation intentionally introduced to, e.g. optimise the codon bias or increase enzyme activity of the encoded protein. The characterisation of the GMM itself includes a description of the new traits conferred by the genetic modification, or those lost in case of gene disruption. Any change in the new organism which may potentially affect its safety should be identified (e.g. altered pathogenicity, or production of toxic compounds). The applicant should also document whether any vector or other sequence not intended for insertion is actually absent in the GMM. This is of special importance when antimicrobial resistance genes are used as selection markers.

4.2. Considerations on the production process

Some aspects of the production and preparation process of the GMM or derived food or food product are also a key in determining product safety, and therefore must be described in detail. In partic- ular, the purification process is one of the essential elements for

egulatory framework governing them.

) No 1332/2008 on food enzymes ) No 1333/2008 on food additives No 1334/2008 on food flavourings

Regulation (EC) No 1829/2003 on GM food and feed

Directive 2001/18/EC on the deliberate release into the environment of GMOs

amins ino acids

– –

reparations Cell extracts –

Breada

Cheesea

Biomass-based food supplementsa

–

Yeast Starter cultures Yogurt

Yeast Starter cultures Yogurt

he appropriate body to provide a correct interpretation of the legislation.

Table 2 Criteria for the categorisation of GMMs and/or their products.

The product contains GMMs The GMMs capable

of replication

The GMMs are in the form The product contains

recombinant genes

The product chemically defined

No No Yes CATEGORY 1

No No No CATEGORY 2

No Yes CATEGORY 3

Yes No No No CATEGORY 2

Yes No No Yes CATEGORY 3

Yes No Yes CATEGORY 4

Yes Yes CATEGORY 4

or rests of them and highly purifiedof spores or VBNC

5J. Aguilera et al. / International Journal of Food Microbiology 167 (2013) 2–7

determining the category to which a product belongs, and therefore, the processes applied will need to be described in detail, and their efficiency validated. If the production strain has been removed, it will be necessary to check whether, in fact, no viable GMMs can be detected in the product. For this, culture-based methodologies are recommended. Samples should be taken from industrial batches of the final product or its concentrate before formulation. Attention should be paid to the possibility that the cells, by effect of the separa- tion process, are metabolically diminished (stressed) and therefore not immediately cultivable, but still viable. This can be done by including non-restrictive cultivation steps for “resuscitation” of the stressed cells, prior to a targeted cultivation in selective media. In case the GMMs are destroyed but not removed, additional data is needed on the possible presence of physically intact cells (inactive but still containing undamaged DNA) or spores when relevant. If the production strain can enter into a viable but non-cultivable state (VBNC), targeted methodologies to detect this kind of cells are suggested (e.g. epifluorescence using specific dyes). In addition, it is essential to test the possible presence of recombinant genes in the final product. For this analysis, the GMM Guidance recommends the use of PCR-based methods, targeting one or more inserted genes. All recombinant genes that may affect the safety (such as those involved in antimicrobial resistance, virulence, or production of toxic com- pounds) should be specifically targeted. The document provides detailed guidance for the design of assays, establishment of limits of detection and adequate controls, including controls for PCR inhibition and lysis of any cells eventually remaining in the sample.

Further detailed information on how to assemble a data set for the evaluation of the genetic modification can be found in a scientific report of EFSA which focused on hypothetical food enzyme produc- tion strains, as examples. These recommendations may also be useful to the preparation of applications for other production strains and derived products (EFSA, 2011b).

4.3. Considerations on the final product

The information requirements with respect to the safety of the product itself depend on the type of product and the legislation under which it falls. The safety assessment of food products consisting, con- taining or derived from GMMs (and therefore falling under Regulation (EC) No 1829/2003) is initially based on the comparative approach. The composition, physical properties and technological properties should be compared between the GMM-derived food and the compar- ator (food produced using the traditional microorganism). The end- points to be compared should be selected on a case-by-case basis, and can include nutrients, antinutrients, organic acids, alcohols, flavour components, density, viscosity, solubility, stability shelf-life, etc. The outcome of the comparative analyses, together with the molecular

and physiological characterisation of the GMM and its newly expressed proteins, constitutes the primary input for the further considerations of the safety of the product.

4.3.1. Toxicology The potential toxicity of the GMM-derived food should be assessed

for any change resulting from the genetic modification. The possible toxicity of the expressed protein(s) can be assessed based on its molec- ular characterisation (sequence, function, biochemical parameters) and its possible similarity to known toxins or bioactive peptides. For the lat- ter, bioinformatic tools should be used to search in available databases. On the other hand, the outcome of the comparative analysis will pro- vide indications on potential toxicity of other constituents of the food. If there are any indications of unintended effects, further toxicological testing should be undertaken with the newly expressed protein(s), the GMM or the final product, depending on the case. For this, the 90-day rodent feeding study is the recommended approach, according to OECD guidelines (OECD, 2012). In cases where the GMM-derived food is largely different from the comparator, or when no appropriate comparator is available, toxicological tests with animals should also be performed. On the contrary, if the results of the molecular and compar- ative analyses reveal no substantial differences or unintended effects of concern, animal feeding studies are not considered necessary.

4.3.2. Allergenicity The risk assessment also considers the potential allergenicity of

the protein(s) newly expressed by the GMM. If the conventional food has already allergenic potential, any possible increase in this potential as a consequence of the use of the GMM should also be eval- uated. Allergenicity is not an intrinsic, fully predictable property of a protein, and there are no validated tests that can establish if a given protein is allergenic. Therefore, a weight of evidence approach should be adopted in the assessment. In this sense, different experimental approaches are suggested in the GMM Guidance, which can be applied on a case-by-case basis. Like the toxicological assessment, bioinformatic searches against relevant databases can identify similarities of the pro- tein expressed by the introduced gene with known allergenic peptides or motifs. Screening for reaction with IgE of sera from sensitised individ- uals may be useful to anticipate whether the protein is likely to be aller- genic. In vitro digestibility tests are also useful to identify proteins that are resistant to pepsin digestion. Other possible sources of evidence are animal tests, in vitro studies with cell cultures, and high throughput techniques such as proteomics.

4.3.3. Nutrition It is also important to evaluate the nutritional impact of the GMM-

derived food. Results of the compositional analysis with respect to the levels of nutrients and antinutrients are the primary source of data.

6 J. Aguilera et al. / International Journal of Food Microbiology 167 (2013) 2–7

If there are differences with respect to the conventional food, or if the comparative approach is not possible, data on the anticipated dietary intake should be provided in order to assess any impact on the nutrition, with particular attention to targeted consumer groups, if relevant.

5. Environmental impact and monitoring

The extent of the ERA linked to the use of the GMM is largely de- pendent on the category to which the product belongs. Provided that there are neither detectable GMMs nor their recombinant genes in the final product, the potential environmental impact resulting from the GMM of products under Categories 1 and 2 is considered negligible, and therefore no further data are required in applications. However, other guidance may be applicable to consider environment-related issues of the product per se, depending on its nature.

When the food contains recombinant genes (Category 3), it is neces- sary to evaluate whether the release of the food harbouring those genes poses any environmental safety concern compared to the release of the conventional food, which does not carry them. The assessment should therefore be based on the possibility that the recombinant genes are horizontally transferred to and expressed in other microorganisms, and on the potential consequences of such expression. The molecular characterisation of the genetic modification provides information on the length and location of the insert and remaining vector sequences, which is helpful to evaluate the potential stability and mobility of the recombinant DNA. Information should also be provided on the environ- ments which could be reached by the GMM-derived food. This should include physical and chemical parameters (pH, temperature, presence of DNA degrading enzymes), and presence of microbial species suscep- tible to natural transformation. In addition, information on the effects of gene expression in terms of selective advantage, pathogenicity or eco- logical alterations will be needed in order to assess the consequences of a theoretical transformation.

The tightest scrutiny corresponds to viable GMMs and products containing them (Category 4). In addition to the assessment of the potential transfer of the recombinant genes, the ERA of these products must cover the possibility that the GMM survives and persists in the en- vironment (including the human gastrointestinal tract), and eventually grows and/or mates with indigenous microorganisms. The assessment should also determine whether the GMM can outcompete natural coun- terparts, and if its relationships with other organisms have been altered as a result of the genetic modification (for example, the development of potential pathogenicity or the loss of symbiotic interactions). Possible alterations of ecological processes such as decomposition or carbon and nitrogen cycles are also to be considered. Therefore, detailed infor- mation on the biology and ecology of the GMM, including habitats, growth parameters, nutrient sources and requirements, production of secondary metabolites, antimicrobial resistance, reproduction, capacity of sporulation, VBNC state, or role in biogeochemical process, is essen- tial for an appropriate evaluation. If the GMM can actively transfer genetic material to other microorganisms, the assessment should also cover the potential effects of the expression of the introduced genes in the receiving organisms. If there are indications of adverse effects of the GMM on indigenous microorganisms or in ecological processes, further data from specifically designed studies should be provided in order to evaluate the consequences of these effects.

Regulation (EC) No 1829/2003 establishes that, in addition to the risk assessment, applications must include a post-market environ- mental monitoring (PMEM) plan according to Directive 2001/18/EC. This request is compulsory for products consisting of or containing GMOs. Therefore, GMM products under Category 4 do require a PMEM. With respect to Category 3, monitoring is only needed if envi- ronmental risks are identified during the assessment. The PMEM has two objectives: first, to evaluate any identified risks and uncertainties and to confirm any assumption made in the ERA, and second, to

identify any adverse effect that could not be anticipated. The first objec- tive is applicable depending on the outcome of the ERA, and involves case-specific monitoring (CSM). The methodology and the endpoints to be studied depend on the specific risks identified, and therefore should be tailored on a case-by-case basis, taking into account the possibility of cumulative and/or long-term effects. The second objective is compulsory for all products under Category 4, and involves general surveillance of the GMM, in order to detect any unforeseen hazard. As general surveillance is not hypothesis-driven, its design and method- ology are challenging. Although there are no established large-scale surveillance and monitoring systems for GMMs, existing monitoring networks could be used whenever possible. In addition, the GMM Guidance recommends the development of focused monitoring sys- tems based on the experience with current surveys for related issues (e.g. pharmaceutical products, consumer's and worker's health).

6. Future

To date, there is no food containing GMMs or derived from GMM biomass commercially available in the EU. Unlike for animal feed, where some applications are currently under assessment at EFSA, this situation is not expected to change in the near future, given the very limited amount of products currently marketed worldwide, and the moderate interest by producers, presumably due to consumer's reticence (EC, 2010). Different factors influence the future of the GMM-derived food in the mid-term. In addition to the ones men- tioned, it is also noteworthy that many products are still in the labora- tory phase, well before reaching an industrial or semi-industrial stage. Funding opportunities for product development, and especially for safety research, will be crucial in determining the moment in which the first applications will be added to the pipeline.

The situation is completely different with respect to GMM-produced food enzymes. These products have been circulating in the European and world markets for decades. The use of gene technologies in the industrial manufacture of fermentation products is at a mature stage, and has pro- foundly impacted the development of this process. Therefore, it is likely that the number of GMM production strains will continue to increase. Until recently, the regulation of food enzymes in the EU existed solely at a national level, and not all Member States have specific legislations regarding the safety of these products. With the entry into force of Reg- ulation (EC) No 1331/2008 and its implementing measures (EC, 2011), all currently marketed products will need to be (re)evaluated, as well as new ones. Within this framework, aimed to provide European con- sumers with the highest standards of food safety, applicants and risk as- sessors can refer to the GMM guidance as a reliable and fit-for-purpose tool for preparing and evaluating applications.

Acknowledgements

The authors thank Antonio Fernandez-Dumont and Elisabeth Waigmann for their critical reading of this manuscript.

References

AMFEP, 2009. List of Commercial Enzymes October 2009. Association of Manufacturers and Formulators of Enzyme Products (Available online (http://www.amfep.org/ content/list-enzymes, February 2013)).

Codex Alimentarius, 2003. Guideline for the Conduct of Food Safety Assessment of Foods Produced Using Recombinant-DNA Microorganisms.

EC, 2001. Directive 2001/18/EC of the European Parliament and of the Council of 12 March 2001 on the deliberate release into the environment of genetically modified organisms and repealing Council Directive 90/220/EEC. Official Journal of the European Communities L106, 1–38.

EC, 2003. Regulation (EC) No 1829/2003 of the European Parliament and of the Council of 22 September 2003 on genetically modified food and feed. OJ L 268, 1–23 (18.10.2003).

EC, 2008a. Regulation (EC) No 1332/2008 of the European Parliament and of the Council of 16 December 2008 on food enzymes and amending Council Directive 83/417/EEC, Council Regulation (EC) No 1493/1999, Directive 2000/13/EC, Council Directive 2001/112/EC and Regulation (EC) No 258/97. OJ L 354, 7–15 (31.12.2008).

7J. Aguilera et al. / International Journal of Food Microbiology 167 (2013) 2–7

EC, 2008b. Regulation (EC) No 1333/2008 of the European Parliament and of the Council of 16 December 2008 on food additives. OJ L 354, 16–33 (31.12.2008).

EC, 2008c. Regulation (EC) No 1334/2008 of the European Parliament and of the Council of 16 December 2008 on flavourings and certain food ingredients with flavouring prop- erties for use in and on foods and amending Council Regulation (EEC) No 1601/91, Regulations (EC) No 2232/96 and (EC) No 110/2008 and Directive 2000/13/EC. OJ L 354, 34–50 (31.12.2008).

EC, 2008d. Regulation (EC) No 1331/2008 of the European Parliament and of the Council of 16 December 2008 establishing a common authorisation procedure for food addi- tives, food enzymes and food flavourings. OJ L 354, 1–6 (31.12.2008).

EC, 2010. Food-related risks. Eurobarometer 73.5. Special Eurobarometer 354. (Available on- line (http://ec.europa.eu/public_opinion/archives/ebs/ebs_354_en.pdf, February 2013)).

EC, 2011. Commission Regulation (EU) No 234/2011 of 10 March 2011 implementing Regulation (EC) No 1331/2008 of the European Parliament and of the Council establishing a common authorisation procedure for food additives, food enzymes and food flavourings. OJ L 64, 15–24 (11.2.2011).

EFSA, 2007. Opinion of the Scientific Committee on a request from EFSA on the intro- duction of a Qualified Presumption of Safety (QPS) approach for assessment of selected microorganisms referred to EFSA. The EFSA Journal 587, 1–16 (2007).

EFSA, 2011a. Scientific Opinion on Guidance on the risk assessment of genetically modi- fied microorganisms and their products intended for food and feed use. EFSA Journal 9 (6), 2193 (2011).

EFSA, 2011b. Trial descriptions of strains of Bacillus licheniformis and Aspergillus niger modified with additional alpha-amylase gene(s). EFSA Journal 9 (6), 2284 (2011).

FDA, 2003. Food and Drug Administration Center for food Safety and Applied Nutrition (CFSAN) Office of Food Additive Safety Agency Response Letter GRAS Notice No. GRN000120. Available online http://www.fda.gov/Food/FoodIngredientsPackaging/ GenerallyRecognizedasSafeGRAS/GRASListings/ucm153936.htm (February 2013).

Franklin, S., Somanchi, A., Wee, J., Rudenko, G., Moseley, J.L., Rakitsky, W., 2011. US patent application No 2011/0293785 A1.

OECD, 2010. Safety Assessment of Transgenic Organisms: OECD Consensus Documents. OECD, Paris (Available online (http://www.oecd.org/document/15/0,3746,en_2649_ 34385_37336335_1_1_1_1,00.html, February 2013).

OECD, 2012. OECD guidelines for the testing of chemicals. OECD, Paris (Available online (http://www.oecd.org/document/40/0,3746,en_2649_34377_37051368_1_1_1_1,00. html, February 2013).

Olempska-Beer, Z.S., Merker, R.I., Ditto, M.D., DiNovi, M.J., 2006. Food-processing enzymes from recombinant microorganisms—a review. Regulatory Toxicology and Pharmacology 45, 144–158.

Pariza, M.W., Johnson, E.A., 2001. Evaluating the safety of microbial enzyme preparations used in food processing: update for a new century. Regulatory Toxicology and Phar- macology 33, 173–186.

Prieto, J.A., Aguilera, J., Randez-Gil, F., 2006. Genetic engineering of baker's yeast: challenges and outlook. In: Shetty, K., Pometto, A., Paliyath, G. (Eds.), Food Biotechnology: Second Edition, Revised and Expanded. CRC Press, Boca Raton, pp. 245–279.